Acetylcholinesterase of Aphis citricola: Properties and significance in determining toxicity of systemic organophosphorus and carbamate compounds

In apterous adults of the spirea aphid, Aphis citricola van der Goot, the optimum conditions for determining acetylcholinesterase (AChE) activity consist of reaction mixture of 0.1 M phosphate buffer (pH 7.5), 10^?3M acetylthiocholine (ASCh), and enzyme extract equivalent to 80 ± 3 μg protein incubated for 15 min at 30°C. The K_m value for ASCh (6.7 × 10^?5M) was much lower than that of butyrylthiocholine (BuSCh) (1.25 × 10^?2M). The enzyme activity was almost completely inhibited by 10^?6M paraoxon or 10^?5M eserine and was 84% inhibited by 10^?5M BW284C51 (a specific AChE inhibitor). DTNB was found to inhibit the enzyme activity and was therefore added at the end of the reaction. AChE activity of A. citricola was inhibited in vitro and in vivo by dimethoxon > dimethoate, and aldicarb sulfoxide > aldicarb > aldicarb sulfone. The in vivo effect correlates well with the toxicity level of the various toxicants. A neurotoxicity index which combines both mortality and in vivo inhibition of the aphid AChE activity is suggested as a measure for determining the toxicity of organophosphorus and carbamate compounds toward aphids.     

Cholinesterase inhibition by methamidophos and its subsequent reactivation

Methamidophos (O,S-dimethylphosphoramidothioate, Monitor) is an organophosphorus, cholinesterase-inhibiting insecticide. The rate constant (k_i) for inhibiting rat plasma cholinesterase (ChE) was 1.57 ± 0.03 × 10³M^?1 min^?1, for rat erythrocyte ChE was 8.86 ± 1.10 × 10³M^?1 min^?1, and for rat brain ChE was 6.58 ± 0.42 M^?1 min^?1. Brain and plasma cholinesterases spontaneously recovered from over 90% inhibition at 30 min to 50% inhibition in 4 and 14 hr, respectively. Pralidoxime increased the rate of reactivation in vitro. In vivo, rats poisoned with methamidophos exhibited signs of cholinergic stimulation. The LD₅₀ of ip methamidophos in male rats was 15 ± 0.7 mg/kg. Pralidoxime (60 mg/kg) and atropine (10 mg/kg) given with the methamidophos increased the LD₅₀ to 52 ± 4.9 mg/kg and 60 ± 0.4 mg/kg, respectively. In rats given 12.5 mg methamidophos (an LD₂₀), ChE activity was depressed 95 ± 12.5% in plasma, 92 ± 0.6% in stomach, and 88 ± 1% in brain at 1 hr after injection. At 48 hr after injection ChE activity had returned to 60% or more of control values in each of the tissues. Administration of a single dose of 60 mg/kg of pralidoxime along with methamidophos did not increase ChE activities at the times and places it was measured.     

Effects of chlordimeform on rectus abdominis muscle of frog

The effects of chlordimeform on rectus abdominis muscle of frog were investigated. Chlordimeform (10^?3M) caused a slow contraction, and at lower concentration (10^?5–10^?3M) it inhibited the acetylcholine-induced contraction in noncompetitive manner. When chlordimeform was applied to the muscle of Rana catesbiana, K⁺-induced contraction was also inhibited in noncompetitive manner. Whereas it had no effect on caffeine-induced contraction.Chlordimeform-induced contraction was not affected by respective addition of d-tubocurarine (10^?4M), procaine (10^?3M), or eserine (0.3 mM), which results were same as that of K⁺-induced contraction. Chlordimeform, at lower concentration (10^?5–10^?3M), inhibits the acetylcholine- and K⁺-induced contractions probably owing to depression of not only the sensitivity of endplate but also the excitability of cell membrane.     

The action of insecticides on neurosecretory neurons in the stick insect, Carausius morosus

The action of insecticides on the spontaneous electrical activity of neurohemal tissue in the stick insect, Carausius morosus, has been studied using extracellular electrodes. The pyrethroid, permethrin, causes a massive increase in the frequency of the spontaneously generated action potentials at concentrations between 5 × 10^?5 and 5 × 10^?8M. Concentrations as low as 5 × 10^?11M are still effective in producing bursting activity.DDT, at concentrations between 5 × 10^?5M and 5 × 10^?6M, produces an overall increase in activity although the bursting activity is less violent than that shown with permethrin. DDT, 5 × 10^?7M, is ineffective at altering the resting pattern.Carbaryl and coroxon cause a transitory increase in electrical activity at 1 × 10^?4M, but are ineffective at 1 × 10^?5M.It is concluded that insecticides could have a direct effect upon the neurohormonal balance in insects.     

Paraoxon-induced release of β-glucuronidase from rat hepatocytes in vitro

A suspension culture of isolated rat hepatocytes was used to reproduce in vitro the paraoxon-induced release of hepatic β-glucuronidase observed in vivo. After a short latent period, exposure of hepatocytes to paraoxon at 10^?7 to 10^?4M resulted in a typical dose-dependent response, with highest release occurring at 10^?4M paraoxon. With 10^?3M paraoxon, however, response was anomalous with a much-decreased enzyme release. As expected from earlier results in vivo, SV₁-oxon exhibited less effect than paraoxon.     

Binding of mercury compounds to the chloroplasts in relation to the inhibition of the Hill reaction

The binding behavior of mercuric chloride (HgCl₂), phenylmercuric acetate (PMA), and ethylmercuric chloride (EMC) to the spinach chloroplasts in relation to the inhibition of the Hill reaction was studied at pH 6.8 and 7.8 using ²⁰³Hg labeled compounds. The pH of the reaction medium did not influence the amount of mercury binding of the chloroplast at various mercurial concentrations, but it altered the inhibition curve of the Hill reaction. Between 0–1 × 10^?5M the binding of Hg²⁺ and EMC were similar and increased linearly with the concentration, while the binding of PMA was similar to the binding of Hg²⁺ only at a concentration below 4 × 10^?6M and was less when the concentration was above 4 × 10^?6M. However, the inhibition of the Hill reaction by these mercury compounds was quite different; at pH 7.8, the I₅₀ values for Hg²⁺, PMA, and EMC were 5 × 10^?6, 2.5 × 10^?6, and 2.5 × 10^?6M, respectively, while at pH 6.8, these values were 4 × 10^?6, 4 × 10^?5, and 2 × 10^?4M, respectively. The differential block of electron flow by the mercury compounds at pH 6.8 and 7.8 was further confirmed by electron spin resonance study.     

Pyrethroid insecticides: Potent,stereospecific enhancers of mouse brain sodium channel activation

Deltamethrin and NRDC 157, pyrethroid insecticides that produce different poisoning syndromes in mammals, enhanced veratridine-dependent, sodium channel-mediated ²²Na⁺ uptake in mouse brain synaptosomes. Concentrations producing half-maximal enhancement were 2.5 × 10^?8M (deltamethrin) and 2.2 × 10^?7M (NRDC 157). This effect was stereospecific: The nontoxic 1S enantiomers had no significant effect on veratridine-dependent activation. At high deltamethrin concentrations, enhancement was maximal at 5 × 10^?5?1 × 10^?4M veratridine. Pyrethroid enhancement was completely blocked by 5 × 10^?6M tetrodotoxin, and neither pyrethroid affected ²²Na⁺ uptake in the absence of veratridine at concentrations up to 1 × 10^?5M. The relative potencies of deltamethrin and NRDC 157 in the synaptosomal sodium channel assay agree well with their relative acute toxicities to mice when administered by intracerebral injection. These findings demonstrate that pyrethroids exemplifying both characteristic poisoning syndromes are potent, stereospecific modifiers of sodium channel function in mammalian brain.     

Action of avermectin B1a on the leg muscles and the nervous system of the American cockroach

The action of avermectin was studied in the leg muscle and the central nervous system of the American cockroach, Periplanata americana L. Avermectin at a low concentration (10^?7M) causes a failure of the leg muscles to respond to external stimuli within 30 min without affecting the magnitude of contraction. Avermectin was found to stimulate Cl^? uptake by the leg muscles within 4 min at 10^?7M. The threshold concentration to cause such stimulation was on the order of 10^?8M. This stimulatory action could be antagonized by picrotoxinin (10^?4M) and to a lesser extent by bicuculline methiodide (10^?4M). The phenomenon is observable under both Na⁺-free and K⁺-free conditions. It was concluded that the action of avermectin is to open the chloride channel on the plasma membrane. This action of avermectin does not seem to be mediated through GABA, GABA receptors, diazepine receptors, or picrotoxinin receptor in this insect species, and therefore suggests that avermectin directly attacks the chloride channel proper both in the central nervous and the neuromuscular systems.     

The effects of pyrethroids on the electrical activity of neurosecretory cells from the brain of Rhodnius prolixus

The effects of pyrethroids on the on-going electrical activity of the axons of neurosecretory cells from the brain of fifth instar Rhodnius prolixus have been studied using extracellular electrodes. Low concentrations of the pyrethroids decamethrin, bioresmethrin, permethrin, and bioallethrin all produce dramatic increases in the overall frequency and dramatic changes in the pattern of electrical activity when applied directly to the exposed brain and corpora cardiaca in an otherwise intact insect. This change in activity was brought about by a recruitment in active units and the production of phasic acivity. A doubling of frequency over that of controls was brought about by low doses of the pyrethroids, namely decamethrin, 1 × 10^?10M; bioresmethrin, 2 × 10^?10M; permethrin, 1 × 10^?9M; and bioallethrin, 2 × 10^?7M. Similar hyperactivity of this system occurred during intoxication of intact insects following topical application of LD₉₅ bioresmethrin. The enhanced sensitivity shown by neurosecretory cells over that of other cell types is discussed, as is the possibility that the increases in electrical activity of neurosecretory axons may result in massive neurohormonal release and thereby contribute to the eventual poisoning of the insect.     

Interference of benomyl and methyl-2-benzimidazolecarbamate (MBC) with DNA metabolism and nuclear divisions in Fusarium oxysporum

Methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate (benomyl) severely decreased DNA synthesis when applied at 3.5 × 10^?6M during the G1 phase of germinating conidia of Fusarium oxysporum; nuclear divisions were completely inhibited at a fungicide concentration of 10 × 10^?6M. The same concentration applied only after the S phase also completely inhibited the nuclear divisions. This dual interference of benomyl with DNA formation and mitosis might be related to a disturbed phosphorus metabolism.     

Effects of herbicidal carbamates on mitochondria and chloroplasts

At concentrations near 2 × 10^?4M, barban, chlorpropham, and phenmedipham are inhibitors of the electron transfer in potato and mung bean mitochondria. The inhibition seems to be localized in the flavoprotein region. It affects preferentially the exogenous NADH dehydrogenation, in potato mitochondria (I₅₀, 10^?4M). Succinate dehydrogenation is less inhibited. At noninhibiting concentrations, the studied carbamates cannot uncouple the oxidative phosphorylations. Photosynthesis is completely inhibited by 2.10^?7M phenmedipham, 5 × 10^?5M barban, and 2 × 10^?4M chlorpropham. The inhibition takes place at the PS II level. Moreover, barban and chlorpropham are uncouplers of the photophosphorylations for concentrations between 5 × 10^?5 and 5 × 10^?4M. The effects observed on mitochondrial respiration can also be found on respiration of Acer cultured cells. The effects on isolated chloroplast photosynthesis are also observed for slightly higher concentrations on cultured Chlorella and on pea and oat leaf fragments.     

Herbicide and inhibitor effects on cell leakage in suspension cultures of tobacco (Nicotiana tabacum)

Effects of selected herbicides and respiratory inhibitors on leakage from tobacco (Nicotiana tabacum) cell suspension cultures were studied. Leakage was monitored by quantitation of fluorescein dye released from preloaded cells and by measuring conductivity changes in the suspension medium. The herbicides ioxynil, Barban, 2,4,5-T, MCPB, and PCP (10^?6 to 10^?4M) caused leakage of fluorescein dye and electrolytes within 2 hr of exposure to the herbicides. Potassium cyanide and 2,4-DNP caused appreciable leakage at the same concentrations. Similar responses were induced by anaerobiosis. Atrazine, metolachlor, paraquat, and nitrofen did not induce leakage when tested at concentrations of 10^?6 to 10^?4M.     

Promotion of norepinephrine release and inhibition of calcium uptake by pyrethroids in rat brain synaptosomes

A series of 25 pyrethroids were assessed for their effects on Na⁺-dependent norepinephrine release and on Ca²⁺ uptake in vitro using a crude rat brain synaptosomal preparation. The most effective pyrethroids required a concentration of 3–10 μM to promote norepinephrine release. Plotting release data versus lipophilicity (as log P) for each compound resulted in a parabolic curve with log P_opt being 5.4 for maximal release. The release promoted by most of the compounds assessed at 30 μM could not be or was only partially reversed by either tetrodotoxin or substituting choline for Na⁺ conditions which readily reversed the release promoting effects of veratridine. Thus, many pyrethroids, particularly those without the α-cyano group, did not display their expected effects on the Na⁺ channel in rat brain. When assessed at 5 μM, pyrethroids inhibited, had no effect, or caused increases in the amount of Ca²⁺ incorporated in the presence of ATP. The effectiveness of the various pyrethroids to inhibit Ca²⁺ uptake again displayed a parabolic relationship with log P_opt being 6.4. It was concluded that the variations in pyrethroid effects on norepinephrine release and Ca²⁺ uptake are not solely related to their particular chemical structures, but to lipophilicity. The effects of many pyrethroids on Ca²⁺ metabolism, particularly displacement of bound Ca²⁺, better explain the transmitter release promoting properties in vitro rather than a direct effect on the Na⁺ channel. No direct relationship between known toxicity to mammals and Ca²⁺ inhibition by pyrethroids was established.     

Effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on photosynthesis and respiration of Nitella dactyl cells

Long-term experiments with dactyl cells of Nitella flexilis showed that the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) at a concentration of 1 × 10^?5M affected not only O₂ evolution in the light but also O₂ uptake in the dark. The inhibition of O₂ production was transitory, but dark respiration did not recover. DCMU induced the formation of giant mitochondria which disappeared before cell death. It was concluded that the algicidic effect of 1 × 10^?5M DCMU on N. flexilis, but not necessarily the elongation of mitochondria, was due to the inhibition of mitochondrial respiration and not of photosynthesis.     

Challenging the larvae of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> and assessing the immune responses to nematode-bacterium complex

Helicoverpa armigera is a strong insecticidal resistance developed insect pest. The understanding of its innate immune responses to emerging biocontrol agent entomopathogenic nematode-bacterial complex can provide an opportunity to control this insect in an environmentally benign manner. Study was focused on role of hemocytes changes and PO activity in Steinernema abbasi-Xenorhabdus indica challenged larvae of H. armigera over the time. Total cell count changed effectively from 10.2?±?1.81?×?10⁵ to 15.5?±?3.3?×?10⁵ cells/mm³ upto 9 h and reduced distinctly up to 8.0?±?2.49?×?10⁵ cells/ mm³ in 24 h. PO activity inclined significantly and was recorded highest at 9 h (24.67?±?1.08?×?10² units) and lowest at 24 h (14.34?±?0.74?×?10² units) in total hemolymph with a similar pattern in plasma and the cellular fraction. Phenoloxidase activity in total and cellular component of hemolymph was positively correlated with prohemocytes, granulocytes and oenocytoids. Study showed the hemocytes and PO accounted as active immune responses against nematode infection. The results provide the first insight to understand the hemolytic activity, quick immunosuppression responses of S. abbasi-X. indica and vulnerability of H. armigera.     

Modification of frescon action in Lymnaea stagnalis smooth and cross-striated muscles by agents that alter potential-dependent or receptor-operated calcium permeability

The calcium entry blockers sodium nitroprusside (10^?2M) and sodium pentobarbital (5 × 10^?3M) had different effects on high-potassium-induced and Frescon-induced contractures in a smooth and a cross-striated muscle of the snail Lymnaea stagnalis. This suggests that Frescon causes an increase in sarcolemmal calcium permeability by a mechanism which is different from that utilized by high extracellular potassium.     

The biochemical mechanism of action of the dinitroaniline herbicide oryzalin

Dinitroaniline herbicides such as oryzalin (3,5-dinitro-N⁴,N⁴-dipropylsulfanilamide) disrupt mitosis in the meristematic cells of seedling plants by inhibiting the formation of microtubules. For further understanding of the biochemical mechanism of action of oryzalin, laboratory analyses with isolated plant tubulin must be employed. Plant tubulin from flagella of the alga Chlamydomonas was isolated and purified. This tubulin was incubated with [¹⁴C]oryzalin, and free oryzalin was separated from oryzalin bound to plant tubulin by miniature DEAE-cellulose chromatography. Scatchard analysis predicts a molar ratio of oryzalin bound to plant tubulin of 1.0 ± 0.1 when oryzalin is incubated with plant tubulin for 30 min at pH 6.9 and 25°C. The association constant for the oryzalin-tubulin complex is 2.08 ± 0.08 × 10⁵M^?1 at 25°C. The thermodynamic values for the formation of the oryzalin-tubulin complex at 25°C are ΔG^o = ?7.25 ± 0.02 kcal mol^?1, ΔH^o = 6.5 ± 0.2 kcal mol^?1, and ΔS^o = 46 ± 2 cal mol^?1 deg^?1 (mean ± standard error). Oryzalin has little or no affinity for intact microtubules, previously denatured plant tubulin, actin, bovine serum albumin, calmodulin, ferredoxin, trypsin, or urease, indicating oryzalin is specific for the biologically active conformation of plant tubulin. Oryzalin binds to plant tubulin to form a complex that may be incapable of polymerizing into microtubules.     

Trifluralin effects on carrot callus tissue

The effect of trifluralin on the growth, oxygen uptake, and adenosine phosphates level of carrot (Daucus carota L.) callus tissue was determined over a period of 18 days after subculture. The herbicide (10^?4 and 10^?5M) reduced fresh weight gain significantly; the reduction was less with lower trifluralin concentrations. Dry weight accumulation was not inhibited until after Day 6 of the test period and thereafter was reduced by all concentrations tested. Oxygen consumption was inhibited by trifluralin (10^?4 and 10^?5M) throughout the test period. Concentrations of 10^?6.10^?7, and 10^?8M produced variable effects. The response profile of O₂ consumption in the presence of dinitrophenol was different from that of trifluralin. Analysis of adenosine phosphates level gave no clear response trend. Energy charge values of 0.7 to 0.85 were obtained for untreated tissue. Trifluralin had no effect on energy charge until Day 9 (10^?4M) and after Day 15 (all concentrations).     

Cartap activity on the cockroach nervous and neuromuscular transmission

By employing intracellular electrodes on the 6th abdominal ganglion, Cartap hydrochloride 10^?5M caused in all experiments a block of the provoked stimulus transmission and a decrease of the cell membrane resting potential; the giant fiber conduction was not affected. In the experiments with extracellular electrodes Cartap 10^?5M provoked a marked increase of the spontaneous activity followed by block which partially disappeared after washing. The same effects were obtained on spontaneous activity when Cartap 10^?5M was used on the denervated 6th abdominal ganglion and in experiments conducted at 0 Ca²⁺ or at 20 mM Mg²⁺. Cartap 10^?5M did not affect the response to direct or indirect stimulation of cockroach neuromuscular preparation. These results tend to confirm that Cartap affects the postsynaptic region of the ganglionic nervous junction. The possible cause of the resting potential decrease is also discussed.     

Influence of monuron on photosystem II and light-dependent 14CO2 fixation in isolated cells of C3 and C4 plants

Cells were isolated from the developing leaves of Ipomoea aquatica (water spinach), a C₃ plant, and three kinds of C₄ plants, namely, Digitaria sanguinalis (NADP⁺-specific malate dehydrogenase type), Panicum miliaceum (NAD⁺-specific malic enzyme type), and Panicum texanum (phosphoenopyruvate carboxy kinase type), to study the effect of monuron on light-dependent ¹⁴CO₂ fixation and oxygen evolution. Bundle sheath cells, except for those of D. sanguinalis, and mesophyll cells of all plants fixed approximately the same amount of ¹⁴CO₂. Monuron, at the range used (2 to 10 × 10^?7M), showed strong inhibition in the mesophyll cells of water spinach and in bundle sheath cells of P. miliaceum and P. texanum and moderate inhibition in the mesophyll cells of all C₄ plants. In the bundle sheath cells of D. sanguinalis the low rate of ¹⁴CO₂ fixation was stimulated to some extent by the addition of malate and ribose 5-phosphate. The I₅₀ value was 6 × 10^?7M for the sensitive cells. Monuron inhibited the oxygen evolution of all seven cell types and their I₅₀ values varied between 3 × 10^?7 to 6 × 10^?7M. The differential response of isolated plant cells from different species to light-dependent CO₂ fixation in the presence of monuron may also be involved in urea herbicide selectivity and undoubtedly is due to the differential photosynthetic pathways present nn them.